CAdA and CdG, which we previously reported, suppressed the production of a wild type HBV genotype Ce (HBVWTCe) in the HBVWTCe plasmid-transfected Huh7 cells and potently decreased the HBVWTCe viremia level in HBVWTCe-exposed human-liver-chimeric-mice. CAdA and CdG were also potent against a highly ETV-resistant HBV variant containing three critical amino acid substitutions: L180M, S202G, and M204V (HBVL180M/S202G/M204V) as examined in HBVL180M/S202G/M204V-plasmid-transfected Huh7 cells and HBVL180M/S202G/M204V-exposed human-liver-chimeric mice. However, both compounds proved to be greatly more cytotoxic than ETV and they were dropped from further development. We, thus, based on the structural findings we had obtained, continued the design and synthesis of approximately 220 novel nucleoside analogs containing a cyano moiety at the 4'-position of the ribose and identified CMCdG. It is of note that CMCdG structurally resembles CdG in that both compounds have 4'-cyano moiety and also resembles ETV in that both of CMCdG and ETV have an exocyclic double bond or methylidene in the 4' position of the cyclopentyl moiety. To determine whether CMCdG reduces the amount of intracellular HBVWTD DNA in HepG2.2.15 cells, DNA isolated from HepG2.2.15 cells, which were cultured in the presence of CMCdG over 14 days, was subjected to real-time HBV-PCR. Antiviral activity was scored as the percent inhibition relative to that of drug-unexposed control cells. The activity of CMCdG against HBVWTD was significantly more potent than ADV and TDF and slightly more potent than TAF, although that of CMCdG was significantly less potent than that of ETV or CdG. The anti-HIV activity of CMCdG and other agents was also examined. ETV was least potent against HIV-1 as previously described. TDF, TAF, and CdG were highly potent against HIV-1 with 50% inhibitory concentration (IC50) values ranging from 0.4 to 36 nM, while ADV was found to be only moderately active against the wild-type HIV-1LAI. CMCdG was also moderately active against HIV-1LAI with an IC50 value of 850 nM. Importantly, unlike CdG that is the 4'-cyano moiety-containing prototypic nucleoside reverse transcriptase inhibitor for CMCdG, CMCdG was almost virtually void of cytotoxicity in HepG2.2.15, Huh7, PXB, and MT-2 cells with 50% cytotoxicity concentration (CC50) values of 500 uM and in HepG2 cells with CC50 value of 89 uM. Thus, the selectivity index (S.I.) of CMCdG in relation to its activity against HBVWTD resulted in greater than 21,739, which was much greater than those of ADV, TDF, TAF, and CdG. Each S.I. of ETV and CMCdG could not be compared since the use of concentrations greater than 500 uM was not practically feasible for the determination of the exact CC50 values. We further examined the effects of CMCdG on cellular mitochondrial DNA (mtDNA), cell growth, and cell viability using two cell lines, MOLT-4 and Daudi. After 7 days of culture in the presence of various concentrations of a test agent, the cells were harvested and subjected to determination of cell growth, cell viability and DNA extraction (for real-time mtDNA-PCR). In the case of ETV, a sharp decline in cell viability and growth was seen at 10 uM in MOLT-4 cells and the same was seen at 100 uM in Daudi cells. There was also a sharp reduction in the amount of mtDNA at 10 and 100 uM in MOLT-4 and Daudi cells, respectively. By contrast, there was no sharp reduction when the cells were cultivated in the presence of CMCdG in neither of MOLT-4 or Daudi cells. There was only a slight reduction in cell viability, cell growth, and the amount of mtDNA at 100 uM. We subsequently asked whether CMCdG blocked the replication of HBVWTCe, HBVETV-RL180M/S202G/M204V, and HBVADV-RA181T/N236T. ETV effectively reduced the synthesis of HBVWTCe at 1 and 102 nM concentrations giving an IC50 value of 7 nM. However, as expected, ETV at 1 to 105 nM concentrations failed to effectively block the DNA synthesis of HBVETV-RL180M/S202G/M204V even at the highest concentration (105 nM), giving ETV's IC50 value of 53,090 nM, although ETV fairly well blocked the synthesis of HBVADV-RA181T/N236T DNA, giving an IC50 value of 184 nM. ADV moderately well blocked the synthesis of HBVWTCe and HBVETV-RL180M/S202G/M204V DNA, giving IC50 values of 1,495 nM and 18,071 nM respectively, while it failed to block the synthesis of HBVADV-RA181T/N236T DNA, giving an IC50 value of 48,905 nM. TDF quite well suppressed the synthesis of HBVWTCe, HBVETV-RL180M/S202G/M204V, and HBVADV-RA181T/N236T, giving IC50 values of 256 nM, 521 nM, and 228 nM, respectively. CMCdG also effectively blocked the synthesis of HBVWTCe and HBVADV-RA181T/N236T DNA with the IC50 values of 252 and 150 nM, respectively, and it fairly well blocked the DNA synthesis of HBVETV-RL180M/S202G/M204V, giving an IC50 value of 3,558 nM. We then asked whether CMCdG blocked the replication of HBV in hu-liver-chimeric mice. In 8 weeks following the inoculation of such mice with HBVWTCe, they were orally gavaged with ETV or CMCdG (prepared at a concentration of 0.1 mg/mL in saline) using oral sondes so that the dose administered resulted in 1 mg/kg/day. Just before the administration of ETV, HBV copy numbers in their plasma were as high as 9 x 108/ml; however, the viremia levels significantly went down by day 7 of ETV administration and further viremia reduction occurred even after the termination of ETV administration to the lowest (by day 21). CMCdG comparably blocked the replication of HBVWTCe under the same conditions (p=0.10). In 8 weeks following the inoculation of hu-liver-chimeric mice with an HBVETV-RL180M/S202G/M204V, the viremia levels had reached 7x107 to 2x108/ml. However, ETV, at a dose of 1 mg/kg/day, showed essentially no reduction in the HBVETV-RL180M/S202G/M204V viremia levels. By contrast, CMCdG at the same dose of 1 mg/kg/day, brought about a significant level of viremia reduction by day 7 of administration. The greatest magnitude of viremia reduction with CMCdG was 1.1 log10 copies/ml (average of 1.2 and 1.0 log10 copies/ml). Over the 14-day period of administration, the HBVETV-RL180M/S202G/M204V viremia reduction in CMCdG-receiving mice was significantly lower compared to that in ETV-receiving mice (p0.0001). However, CMCdG had no impact on the levels of HBsAg in their sera during the period of CMCdG administration in either of HBVWTCe- or HBVETV-RL180M/S202G/M204V-exposed mice, suggesting that CMCdG reduced viremia by inhibiting HBV's reverse transcriptase activity, but did not block the transcription of cccDNA into mRNA or the translation to produce HBV proteins. CMCdG caused no significant changes in body-weights and serum human albumin levels in mice. Taken together, the present data on CMCdG warrant further studies toward the clinical development as a potential therapeutic for infection with wild-type and/or drug-resistant HBV and should shed light to further optimization and development of more potent and less toxic anti-HBV therapeutics with high genetic barrier to the emergence of drug-resistant HBV variants.